Dual functions of microRNA-17 in maintaining cartilage homeostasis and protection against osteoarthritis

Damaged hyaline cartilage has no capacity for self-healing, making osteoarthritis (OA) “difficult-to-treat”. Cartilage destruction is central to OA patho-etiology and is mediated by matrix degrading enzymes. Here we report decreased expression of miR-17 in osteoarthritic chondrocytes and its deficiency contributes to OA progression. Supplementation of exogenous miR-17 or its endogenous induction by growth differentiation factor 5, effectively prevented OA by simultaneously targeting pathological catabolic factors including matrix metallopeptidase-3/13 (MMP3/13), aggrecanase-2 (ADAMTS5), and nitric oxide synthase-2 (NOS2). Single-cell RNA sequencing of hyaline cartilage revealed two distinct superficial chondrocyte populations (C1/C2). C1 expressed physiological catabolic factors including MMP2, and C2 carries synovial features, together with C3 in the middle zone. MiR-17 is highly expressed in both superficial and middle chondrocytes under physiological conditions, and maintains the physiological catabolic and anabolic balance potentially by restricting HIF-1α signaling. Together, this study identified dual functions of miR-17 in maintaining cartilage homeostasis and prevention of OA.

O steoarthritis (OA) is the most prevalent degenerative disorder of synovial joints and leads to chronic disability due to pain and associated joint dysfunction 1 . Currently, no treatments can effectively impede the progression of OA 2 , and surgical joint replacement is ultimately required in many affected patients 3 . The structural integrity of healthy cartilage is maintained by a fine-tuned balance between anabolism and catabolism of extracellular matrix (ECM) components in chondrocytes 4 . Chondrocytes display zone-specific heterogeneity amongst different layers of the articular cartilage 5,6 . However, during OA pathogenesis, the cartilage matrix is gradually degraded owing to excessive expression of multiple matrix-destructive enzymes, particularly matrix metallopeptidases MMP3, MMP13, and aggrecanase-2 (ADAMTS5), produced by chondrocytes in response to biochemical and biomechanical stimuli 7 . Thus, exploring molecular mechanisms underlying the imbalance between anabolic and catabolic pathways is expected to help identify new therapeutic targets for OA.
MicroRNAs (miRNAs) had been reported to modulate various pathological processes during OA by repressing their target genes. Supplementation of those reported individual miRNAs has been shown to elicit detectable levels of protection against OA, yet without robust repair [8][9][10] . Targets of those reported miRNAs were almost always limited to single catabolic factors, while a promising therapeutic option for OA may require concomitant inhibition of multiple matrix-degrading enzymes. Perhaps a combinatorial usage of multiple miRNAs is an option, unless one microRNA can be identified to simultaneously target multiple, if not all of the key OA-associated matrix-degrading enzymes.
MiR-17-5p (miR-17) belongs to the miR-17~92 cluster 11 , and dysregulation of this microRNA cluster has been found to be associated with skeletal malformation and related growth defects in humans 12 . In a published study by Han et al. through serial mutations of each of the 4 seed family members within this cluster, only deletion of miR-17 seed family resulted in vertebral transformations and shortening of the fifth mesophalanx, indicating that miR-17 has a profound role in controlling skeletal development 13 . However, the function of the miR-17~92 cluster, especially miR-17, has not been fully elucidated in adult cartilage maintenance and OA progression. The miR-17~92 cluster could be regulated by BMP family proteins including BMP2/4 during cardiac development 14 . Another BMP family member, growth differentiation factor 5 (GDF-5) plays key roles during joint morphogenesis 15 . Functional single-nucleotide polymorphisms linked to reduced Gdf5 gene transcription are currently the most consistent risk factor for adult-onset OA across human populations 16,17 . Interestingly, skeletal defects resulted from dysfunction of miR-17~92 cluster showed shortening of phalangeal elements and bony fusions of the joints 12 , which are also observed with GDF5 deficiency 18 , suggesting an inter-connection between GDF5 and miR- 17. In this study, we used destabilization of the medial meniscus (DMM)-induced mouse OA model, as well as a series of gain-and loss-of-function analyses plus single-cell RNA sequencing (scRNAseq) to examine the regulation and function of miR-17, together with molecular mechanisms underlying OA pathophysiology.

Results
MiR-17 reduction plays a critical role in OA progression. We performed in-silico screening using 30 most-abundantly expressed miRNAs identified in chondrocytes 19 , as well as miRNA target prediction programs to search for miRNAs predicted to simultaneously target MMP13 and ADAMTS5. Such a screen resulted in 4 miRNAs, which are miR-17, miR-20a, miR-140-3p, and miR-27-3p ( Supplementary Fig. 1a). MiR-140-3p and miR- 27-3p have been reported to play essential roles in cartilage development and homeostasis 20,21 . Interestingly, miR-17 and miR-20a happened to be located in the miR-17~92 cluster. The miR-17~92 cluster contained a total of 6 microRNA members including the miR-17 family (miR-17 and miR-20a), the miR-18 family (miR-18a), the miR-19 family (miR-19a-3p and miR-19b-3p), and miR-92a-3p. We investigated a possible relationship between GDF-5 and miR-17~92 cluster, and found that miR-17 was the most highly expressed and strongly upregulated miRNA within this cluster, in response to GDF-5. MiR-20a was also significantly upregulated by GDF-5 ( Supplementary Fig. 1b). Since miR-17 shared the same seed sequence with miR-20a and had a higher expression level, we selected miR-17 for further study.
In uninjured cartilage, miR-17 expressing cells were mainly located in the superficial and transitional (middle) zones, accounting for 31.32% and 64.91% respectively, of all miR-17 positive cells in the cartilage as detected by fluorescence in situ hybridization (FISH) (Fig. 1a, Supplementary Fig. 1c). In DMMinduced OA mice, Osteoarthritis Research Society International (OARSI) grade scores increased overtime, and percentages of miR-17-positive cells over total cells in the cartilage declined from 41.99% to 8.03%, by week 8 and on-ward ( Fig. 1a, b). At 4 weeks after DMM surgery, the percentages of miR-17-positive cells dropped from 70.39% over total DAPI + cells within the superficial layer to 30.83%, and from 65.75% to 36.1% in the middle layer ( Supplementary Fig. 1d). qRT-PCR also demonstrated consistent decreases of miR-17 expression overtime after surgery ( Supplementary Fig. 1e), consistent with previous report 22 . To explore the role of miR-17 reduction in OA progression, we injected antagomir-17 at dose of 3 nmol intraarticularly to neutralize the endogenous miR-17 ( Supplementary  Fig. 1f). This administration indeed aggravated the cartilage destruction and increased OARSI scores at 4 weeks post DMM surgery (Fig. 1c). In sham-operated mice, however, joints received antagomir-17 injection still manifested normal cartilage architecture (Fig. 1c), suggesting inhibition of miR-17, per se, is not sufficient to cause cartilage damage. For gain-of-function study, injection of 1.5 nmol agomir-17 into DMM-induced joints weekly, starting at 4 weeks post DMM surgery, significantly increased miR-17 expression as well as numbers of miR-17positive cells, along with improved OARSI scores, after 4 injections (Fig. 1d, Supplementary Fig. 1g, h). In shamoperated mice, agomir-17, like antagomir-17, also had no influence on the cartilage structure (Fig. 1d), suggesting endogenous levels of miR-17 are already suffice and excessive miR-17 does not elicit more protection, yet it does not lead to harmful outcome either.
Genetically, the miR-17 gene is located in the miR-17~92 polycistronic cluster. We generated conditional knockout (cKO) mice with chondrocyte-specific miR-17~92 deletion, showing reduction of miR-17 levels ( Supplementary Fig. 1i). Immediately following tamoxifen treatment, DMM surgery was performed on both control and cKO mice (8 weeks old). Four weeks later, cKO mice already developed OA with extensive loss of cartilage and higher OARSI scores, whereas control DMM mice had only shown mild cartilage degradation without showing significantly higher OARSI scores compared to non-injury controls (Fig. 1a, e). It was obvious that in DMM joints, deletion of miR-17~92 cluster resulted in greater severity of cartilage destruction than antagomir-17 injection. qRT-PCR indicated that expression levels of miR-17 in cartilage were substantially higher than those of the other five members, while miR-19b-3p (miR-19) and miR-92-3p (miR-92) were also highly expressed ( Supplementary Fig. 1j). In order to investigate the function of individual miRNA within the miR-17~92 cluster in OA progression, we performed rescue experiments by injecting each miRNA (miR-17, miR-19 and miR-92) into joints of cKO mice. As expected, supplementation with agomir-17 from 1 week after DMM surgery in cKO mice, rescued cartilage destruction and improved OARSI scores by 4 weeks post-surgery (Fig. 1f). Agomir-19 did not elicit significant cartilage protection, while agomir-92 demonstrated certain degrees of protection, but less than that of agomir-17 (Supplementary Fig. 1k), which further substantiate the role of miR-17 in protection against OA. Aging is a well-known prominent risk factor for OA 23 , we analyzed miR-17 expression in 3-and 12month-old mice and found decreased miR-17 expression with aging ( Supplementary Fig. 2a). After DMM surgery, aged (12month-old) mice showed more extensive cartilage destruction ( Supplementary Fig. 2b). Strikingly, agomir-17 was still effective in protecting aged DMM mice ( Supplementary Fig. 2c).
miR-17 suppresses cartilage destruction by inhibiting multiple catabolic factors. To explore mechanisms by which miR-17 inhibits cartilage destruction during OA progression, we used multiple microRNA target prediction programs. Nine potential targets relevant to catabolism in OA cartilage were selected, including Epas1 (encoding HIF-2α), Mmp2, Mmp3, Mmp13, Mmp24, Adamts5, Adamtsl2, Adam9 and Nos2 (nitric oxide synthase-2), in addition to previously published targets 24,25 . We examined expression patterns of these genes in OA chondrocytes and data indicated that 5 genes (Epas1, Mmp3, Mmp13, Adamts5, and Nos2) were elevated significantly in IL-1β-treated chondrocytes as well as DMM cartilage ( Supplementary Fig. 3a, b). After confirming that Epas1 was not a direct target for miR-17 (Fig. 2a), we selected 3′-UTRs of Mmp3, Mmp13, Adamts5 and Nos2 for further characterization ( Supplementary Fig. 3c). Luciferase reporter assays verified that miR-17 suppressed the expression of all 4 genes through 3′UTR seed recognition sequences, because mutations of which completely eradicated miR-17-induced suppression (Fig. 2a). In cultured mouse chondrocytes, exposure to proinflammatory factor IL-1β resulted in significant upregulation of all 4 catabolic factors (MMP3,   The injections were preformed weekly for 3 times beginning at 1 week after surgery. n = 6 mice per group. Data are presented as boxplots. Boxplots: center line, median; box limits, 25 to 75th percentiles; whiskers, min to max. NS not significant, *P < 0.05, **P < 0.01, and ***P < 0.001. One-way ANOVA (nonparametric) with Dunn's multiple comparisons test for OARSI grade in (b, c); Mann-Whitney U test (two sided) for (d, e, f); one-way ANOVA with Bonferroni's test for percentage of positive cells in (b). All scale bars, 100 μm. All the analyses were sourced from tibial cartilage. Source data are provided as a Source Data file. MMP13, ADAMTS5 and NOS2), which could be partially reversed by transfection of miR-17 mimic (Fig. 2b). However, SOX9 and COL2A1, which were suppressed by IL-1β, could not be reversed by miR-17 ( Fig. 2b) Fig. 4). In DMM cartilage, in vivo, application of agomir-17 was found to suppress all 4 catabolic factors at mRNA levels ( Fig. 2c). Moreover, numbers of MMP13 + , ADAMTS5 + or NOS2 + cells were also decreased upon agomir-17 treatment (Fig. 2d). Similar phenotypes were observed in DMM cKO mice and DMM with antagomir-17 injections (Fig. 2e, f, Supplementary Fig. 5). Together, these findings demonstrated that miR-17 protected against cartilage destruction by targeting multiple pathological catabolic factors in OA pathologies.
To explore whether similar levels of protection against OA could be achieved through upregulation of endogenous miR-17, we selected GDF-5 as an inducer for miR-17. In cultured mouse articular chondrocytes, exogenous GDF-5 induced expression of miR-17 under IL-1β stimulation ( Supplementary Fig. 6a). Moreover, GDF-5 markedly suppressed IL-1β-induced expression of MMP3, MMP13, ADAMTS5 and NOS2, in a dose-dependent manner, which could be reversed by miR-17 inhibitor, at both mRNA and protein levels ( Supplementary Fig. 6b, c). More importantly, intra-articular injection of GDF-5 restored miR-17 expression and numbers of miR-17 + cells in DMM cartilage, along with reversed OARSI scores ( Fig. 3a, b, Supplementary  Fig. 6d), while intra-articular administration of antagomir-17 together with GDF-5, largely abrogated the protective effect of GDF-5 ( Fig. 3c-    MiR-17~92 deficiency in chondrocytes triggered cartilage degeneration without DMM. In miR-17~92 cKO mice, noticeable weakness of proteoglycan staining in the weight-bearing area of the femoral condyle was observed in knee joints at 4 weeks after tamoxifen injection even without DMM surgery. By 8 weeks, the affected area had expanded to cover nearly half of the joint surface, extended into the calcified layer ( Fig. 4a, b). The pathology featured a marked decrease in cellularity of the superficial layer (70.1% fewer cells than in control mice at 4 weeks), matrix edema (increased cartilage thickness), and splitting (matrix separation) 26 in middle cartilage ( Fig. 4a, b). Unexpectedly, neither staining of MMP13, ADAMTS5 and NOS2 nor the mRNA levels of the pathological catabolic factors showed significant upregulation in degenerated cartilage of cKO mice without DMM surgery (Fig. 4c, d), indicative of a different degenerative mechanism. Yet, intra-articular administration of agomir-17 for 4 weeks still demonstrated robust protective effect against cartilage degeneration in miR-17~92 cKO mice ( Fig. 4a, b), likely via a mechanism other than inhibition of the pathological catabolic factors.
MiR-17 maintains cartilage homeostasis potentially via restriction of HIF-1α levels. It has been well recognized that the joint cartilage is composed of four distinct layers and chondrocytes showed substantial heterogeneity in cellular morphology, arrangement, and matrix production 27 . However, little is known about the difference at the whole transcriptomic levels among the chondrocyte subsets. Additionally, changes in gene transcription of each chondrocyte subset under OA pathology remain to be fully elucidated. To explore the underlying mechanisms by which miR-17~92 deficiency led to cartilage degeneration and/or how miR-17 reversed the phenotype, we performed scRNA-seq on cells isolated from the medial femoral condyle (Fig. 5a). After unsupervised hierarchical clustering, we identified a cluster with high expression of Sox9, Acan, Comp and Fn as a chondrocyte population ( Supplementary Fig. 7a), which could be subsequently subdivided into three clusters (Chon-drocyte1 (C1), C2 and C3) (Fig. 5b).
Mmp2, Col14a1 and Col22a1, which are reported to be expressed mainly in the superficial zone 5,27,28 , were highly enriched in clusters C1 (Mmp2 and Col14a1) or C2 (Col22a1). In addition, C1 cluster also expressed Serpinf1, Lum and Apod; C2, Clic5, Itga6, and Hbegf; and C3, Cytl1, Clu, Col2a1, and Cilp ( Fig. 5c). Immunofluorescent staining revealed distribution of MMP2, APOD, CLIC5, and COL22A1 on the cartilage surface and CYTL1 expressed only in the middle zone (Fig. 5d). We thus inferred that clusters C1 and C2 representing superficial chondrocytes, while cluster C3 representing middle zonal chondrocytes. Prg4 (lubricin), a known superficial chondrocyte marker gene, was highly expressed in cluster C2 and was unexpectedly expressed also in cluster C3 (Supplementary Fig. 7a). It is likely that the Prg4 mRNA is not translated into proteins or that the protein is degraded in middle zonal chondrocytes. Such a mode of step-wise shutting down of a gene has been reported in embryonic stem cells committed to neuronal fate 29 . By GO analysis, cluster C1 was enriched for GO terms featured in collagen fibril organization (Col3a1), and collagen catabolic process (Mmp2 and Mmp14), suggesting high rates of collagen turnover. Cluster C2 was enriched with genes related to epithelial cell migration, which could be linked to lineages of the synovial lining layer 30 . Cluster C3 expressed genes associated with cartilage development (Comp) and collagen fibril organization (Col2a1, Col11a1) (Fig. 5e). In addition, biological processes of C1 and C3 were concentrated on cartilage-bone morphogenesis, but those of C2 focused on "neural crest cell migration" (Supplementary Fig. 7b). Thus, the superficial zone of cartilage may contain two previously unknown intermingled subpopulations of cells, both of which might be decreased in miR-17~92 cKO mice.
We further analyzed scRNA-seq data from control, cKO, and agomir-17 rescued cKO mice. Consistent with histological analysis (Fig. 4b), the percentage of superficial zone C1 and C2 cells in the harvested osteochondral unit were dramatically reduced in cKO mice. Agomir-17 injection reversed abundances of C1 and C2 subsets to 105.3% and 60.4% of the control values, respectively. However, the abundance of cluster C3 remained relatively unchanged upon miR-17~92 deletion (Fig. 6a). These data suggested that the numbers of C1 and C2 superficial zone cells were more sensitively fine-tuned by levels of miR-17. Gene expression analyses further demonstrated that in all three subpopulations of cells, miR-17 administration could pretty much revert altered gene expression due to miR-17-92 cKO, back to control levels (Fig. 6b). Additional GO analysis of miR-17 dependent genes in all three cell populations included "collagen fibril organization", whereas "ECM organization" and "cartilage a Luciferase activity of 293 T cells co-transfected with dual-luciferase reporter constructs containing wild-type or mutated 3′-UTRs, as well as negative control (mimic NC) or miR-17 mimic. The data presented are mean percentage changes over mimic NC ± s.e.m. n = 6 biologically independent samples (NC + wt UTR, miR-17+wt UTR); n = 4 biologically independent samples (NC + mut UTR, miR-17 + mut UTR). b Mouse articular chondrocytes were transfected with miR-17 mimic (50 nM) or mimic NC and treated with or without IL-1β (5 ng/mL) for 24 h. The protein levels of catabolic and anabolic factors were determined by western blot followed by densitometry analysis. Blots are representative of three independent experiments. c, d qRT-PCR analysis of catabolic genes in knee cartilage (c), and representative images of IHC staining and quantification of MMP13 + , ADAMTS5 + and NOS2 + cells in joint sections (d) of mice subjected to sham or DMM surgery and intra-articular injections of agomir-NC or agomir-17 (1.5 nmol) for 4 weeks, beginning at 4 weeks after surgery. n = 5 biologically independent samples per group in c. For MMP13 and ADAMTS5 staining, n = 4 mice (sham); n = 5 mice (DMM, DMM + agomir-17). For NOS2 staining, n = 4 mice per group. e, f qRT-PCR analysis of catabolic genes (e) and representative images of MMP13, ADAMTS5, and NOS2 immunostaining, and quantification of positive cells (f) from knee joints of cKO mice subjected to DMM surgery and intra-articular injection of agomir-NC, or agomir-17 (2 nmol). The injections were performed at 1 week after surgery and samples were collected at 2.5 weeks after surgery. n = 4 biologically independent samples per group in (e); n = 4 mice per group in (f). All scale bars, 100 μm. Data are presented as mean ± s.e.m. or boxplots (center line, median; box limits, 25 to 75th percentiles; whiskers, min to max), and dots represent individual mice or biologically independent samples. NS, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 in (a, c-f). *P < 0.05 versus control group,°P < 0.05 versus IL-1β + mimic NC in (b). Two-sided Student's t test for (a); one-way ANOVA with Bonferroni's test for (b-f). development" were enriched in C1 and C3 (Fig. 6c). A comprehensive downregulation of collagen expression (Col3a1 in C1; Col1a1 and Col22a1 in C2; Col2a1 in C3) was observed in cKO mice ( Supplementary Fig. 8), which was validated by immunohistochemical analyses (Supplementary Fig. 9). In accordance with this, human OA cartilage samples demonstrated loss of COL1A1 in cartilage surface and decreased COL2A1 expression in undamaged region, which may represent early OA pathology ( Supplementary Fig. 10). Importantly, miR-17 expression was also significantly reduced in human OA cartilage (Fig. 6d), indicative of the human relevance of our findings.
We mapped the expression patterns of both anabolic and catabolic genes in all three clusters (Supplementary Fig. 8). Normal C1 chondrocytes expressed high levels of Mmp2 and Mmp14; but Mmp3, Mmp13 and Adamts5 were not highly expressed in three clusters. With deletion of miR-17~92, without DMM, the expression of these catabolic genes did not increase, instead, Mmp2 and Mmp14 decreased expression. Importantly, expression of anabolic genes including Fgfr1, Bmp2, Acan, Fn1, Col14a1, Prg4, and Loxl2 were also decreased in different cell populations, indicative of reduced anabolic process leading to cartilage degeneration. Intraarticular supplementation of agomir-17, restored the overall balance   between anabolism and catabolism, and prevented cartilage degeneration. In scRNA-seq data, we found that genes for collagen-modifying enzymes were abundantly increased in cKO chondrocyte populations, including those of collagen prolyl hydroxylase (P3h3), lysine hydroxylase (Plod2) and lysyl oxidase (Lox), two of which were inhibited following agomir-17 injection (Fig. 6e). Simultaneously, expressions of genes for collagen were reduced ( Supplementary Fig. 8). Reduced collagen synthesis and increased collagen modifications were also reported previously in growth plate cartilage of Phd2 chonmice with accumulation of the hypoxia-inducible factor HIF-1α 31 , allowing us to speculate that HIF-1α might play critical roles in dysregulation of collagen turnover in miR-17~92 cKO mice. We thus examined expression levels of HIF-1α in joint cartilage of wild type and cKO mice and found that HIF-1α protein levels were elevated in cKO cartilage, along with decreased expression of SOX9, COL2A1, and MMP2. With administration of agomir-17, changes in the expression of HIF-1α, SOX9, COL2A1, and MMP2 were reversed (Fig. 6f). In cultured mouse chondrocytes treated with PHD2 inhibitor (IOX2 at 20 μM) for 72 h, HIF-1α was elevated and levels of P3h3, Plod2 and Lox were increased. While the expression of SOX9 and MMP2 were decreased. Treatment with combination of miR-17 inhibitor and IOX2 caused increased expression of HIF-1α and collagenmodification enzymes, and further decreases in SOX9 and MMP2 expression. In contrast, overexpression of miR-17 resulted in significant decreases of HIF-1α, along with increased expression of SOX9, MMP2 and decreased expression of collagen-modification enzymes (Fig. 6g-i). However, we did not observe significant changes in COL2A1 protein expression upon IOX2 treatment, probably because increased collagen-modification protected COL2A1 from degradation. This is slightly different from the in vivo situation, potentially due to lack of mechanical loading in culture conditions. Given that the target site of miR-17 in 3′UTR of HIF-1α has been well characterized in previous report using miRNA-reporter assays 32 , we conclude that miR-17 maintains cartilage homeostasis potentially through directly targeting HIF-1α, by which collagen turnover was sustained in balance under physiological circumstances.

Discussion
In this study we showed that miR-17, which is highly expressed in superficial and middle zones of the articular cartilage, protected against DMM-induced cartilage destruction by simultaneously targeting pathological catabolic factors, including MMP3, MMP13, ADAMTS5, and NOS2. We also discovered that GDF-5 upregulated endogenous miR-17 expression to exert its protective function against OA. ScRNA-seq analyses of miR-17~92 knockout chondrocytes and further experiments revealed that, miR-17 also positively regulated physiological catabolic and anabolic factors involved in cartilage homeostasis potentially via restriction of HIF-1α accumulation (Fig. 7). The miR-17~92 cluster contains 6 different microRNA members. Together, they target more than 30 genes, which are known to be involved in diseases of the nervous, cardiovascular, and skeletal systems 33 . In the current study, we have demonstrated critical roles of miR-17 in regulating cartilage homeostasis, although we do not exclude the possibility that miR-92, miR-20a, or even miR-19 also participate in the regulation to certain extent. In DMM-induced OA, major catabolic factors including MMP3/ 13, ADAMTS5, NOS2 are upregulated and function to destroy cartilage in the pathological condition. During OA, endogenous miR-17 is downregulated, leading to "loss of protection" of the cartilage. MiR-17, through simultaneously and directly target 3′ UTRs of these genes, effectively suppress the function of these pathological catabolic factors. However, the severity of cartilage destruction in DMM mice with antagomir-17 injections was less than that in DMM cKO mice. Knockdown of endogenous miR-17 by injecting antagomir-17, unlike deletion of the whole miR-17~92 cluster, failed to trigger a spontaneous cartilage degeneration. These results indicated functional cooperation among members of miR-17~92 cluster and they might share similar targets. For example, miR-92 has been reported to directly target ADAMTS5 in chondrocytes 34 , and miR-19 was also predicted to target HIF-1α.
The superficial layer of the articular cartilage is the first to sense physio-pathological stimuli and response to mechanical loadings. ScRNA-seq and subsequent immunohistochemical analyses demonstrated that the superficial zone of the cartilage contained two intermingled cell subpopulations with one carrying features of synovial lining layer expressing type XXII collagen and lubricin (Prg4), and the other expressing types I, III, IV, VI, XIV collagen, etc. These collagens function to reinforce cartilage structural network by crosslinking collagen fibrils, mediating intermolecular interactions and coordinating cell-matrix interactions 35 . Recent report using scRNA-seq of human osteoarthritic cartilage also indicated the presence of chondrocyte subtypes 36 . Together these observations demonstrated that cellular heterogeneity existed not only across different zones but also within a single layer of the cartilage. The superficial zone was found to be a structurally important element, which greatly limits swelling of the entire cartilage layer 37,38 by maintaining fluid pressurization within the tissue 39 . Thus, the decrease of superficial chondrocytes in cKO cartilage and subsequent degeneration of superficial zone contributed to matrix edema, leading to increases of cartilage thickness. Superficial chondrocytes appeared to be more sensitive to   To maintain cartilage homeostasis, a continuously ongoing degradation of old matrix is critical for deposition of newly synthesized matrix molecules 7 . In this study, we found MMP2, 14, and ADAMTS2 were expressed by healthy superficial chondrocytes as physiological catabolic factors, while MMP3, 13, and ADAMTS5 were not involved. In fact, it has been reported that a   p<0.0001 Fig. 6 MiR-17 maintains balanced anabolism and catabolism in subsets of articular chondrocytes. a Cell percentages in each cluster of control, miR-17~92 cKO and cKO + agomir-17 mice. b Genes reversed by miR-17 supplementation in miR-17~92 cKO mice in clusters C1, C2, and C3. c GO analysis of genes upregulated in clusters C1, C2, and C3 in miR-17~92 cKO+ agomir-17 mice in (b). d qRT-PCR analysis of miR-17 expression in normal (n = 6), undamaged (n = 10) osteoarthritic and damaged (n = 18) osteoarthritic human cartilage. e Violin plots showing changes in levels of collagen-modifying genes in control, miR-17~92 cKO and cKO + agomir-17 mice. f Protein levels of HIF-1α, SOX9, COL2A1, and MMP2 in articular cartilage of control, miR-17~92 cKO and cKO + agomir-17 mice at 2 weeks after tamoxifen injections were determined by western blot followed by densitometry analysis. Blots are representative of three independent experiments. g-i Mouse articular chondrocytes were transfected with miR-17 inhibitor (100 nM) or inhibitor NC or miR-17 mimic (50 nM) or mimic NC and treated with or without IOX2 (20 μM) for 72 h. The protein levels of HIF-1α, SOX9, COL2A1 and MMP2 were determined by western blot followed by densitometry analysis. Blots are representative of three independent experiments (g). The gene expression of P3h3, Plod2, and Lox were determined by qRT-PCR. n = 3 biologically independent samples (none-IOX2 treatment groups); n = 4 biologically independent samples (IOX2 treatment groups) (h, i). Data are presented as means ± s.e.m. or boxplots (center line, median; box limits, 25 to 75th percentiles; whiskers, min to max), and dots represent biologically independent samples. *P < 0.05, **P < 0.01, and ***P < 0.001 in (d, h, i); *P < 0.05 versus control group,°P < 0.05 versus cKO group in (f); *P < 0.05 versus none-IOX2 group,°P < 0.05 versus IOX2 with inhibitor/mimic NC group in (g) (one-way ANOVA with Bonferroni's test).  significant amount of MMP2 is expressed in the superficial zone of human normal cartilage 40 . In order to match with their highly anabolic metabolism, superficial chondrocyte undergoes robust catabolism to maintain active matrix turnover 41 . Interestingly, in miR-17-92 cluster cKO mice, cartilage underwent degeneration without upregulation of the aforementioned pathological catabolic factors (MMP3,13, etc.), as revealed by scRNA-seq. Instead, miR-17 deficiency led to reduced expression of the physiological catabolic factors (Mmp2,14) as well as anabolic factors (Fgfr1, Acan, Fn1, Col14a1, and Loxl2), and supplementation of miR-17 reversed these changes. The alteration of cartilage matrix would significantly decrease the load bearing capacity of articular cartilage, further damaging the collagen and proteoglycan structure 42 .
Despite the importance of HIF-1α in ECM synthesis 43 , epiphyseal chondrocytes require PHD2-regulated inactivation of HIF-1α to avoid skeletal dysplasia, since pathological increase of HIF-1α signaling interferes with cellular bioenergetics and biosynthesis 31 . It is unknown whether there is a requirement for restriction of HIF-1α signaling in articular chondrocytes. Our data showed the HIF-1α level was enhanced, yet, anabolic as well as physiological catabolic factors were reduced in cKO chondrocytes as well as by prolonged treatment of PHD2 inhibitor. Overexpression of miR-17 caused decreased levels of HIF-1α and restored physiological catabolic and anabolic balance, showing miR-17 functions as a factor to control HIF-1α levels. The appropriate HIF-1α signaling is vital for chondrocytes metabolism and ECM production. It has been reported that any anomalous subtle alterations in anabolic reactions, as indicated in knockdown of Kdm6b 44 or interruption of TGF-β1 signaling 45 , can result in initiation of homeostasis disruption of articular cartilages. Therefore, miR-17 could function under both physiological and pathological conditions, playing different roles in modulating the anabolic and catabolic activities in maintaining joint cartilage homeostasis as well as repair in OA.
In conclusion, our study revealed dual roles of miR-17 in maintaining cartilage homeostasis and prevention of OA, suggesting that miR-17 could be a potentially drug target for OA. Given that the therapeutic benefits of miRNAs in disease intervention have been increasingly documented in preclinical studies 46 , supplementation of miR-17 might be a potent therapeutic approach for OA as well as prevention of cartilage aging.

Methods
Human specimens. Normal human articular cartilage without arthritis was collected from the knee or hip joints from 6 patients with osteosarcoma or trauma who were undergoing surgery. Arthritic cartilage specimens were obtained from individuals with OA within 2 h after arthroplasty. OA was diagnosed according to the criteria of the American College of Rheumatology. The specimens were processed for qRT-PCR and histological examination. The patients without arthritis (1 male and 5 female) had an average age of 55.5 years. The patients with OA (4 male and 14 female) had an average age of 66.06 years. The collection of discarded human tissues was approved by the Research Ethics Board of Affiliated Hospital of Xuzhou Medical University. Written informed consent was provided by all patients involved in this study. The study design and conduct complied with all relevant regulations regarding the use of human study participants and was conducted in accordance with the criteria set by the Declaration of Helsinki.
Mice. Male C57BL/6J (wild type) mice were obtained from the Shanghai Animal Center and randomized into experimental groups. Mice were housed under ambient temperature of 24 ± 2°C, circulating air, constant humidity of 50 ± 10% and a 12 h:12 h light/dark cycle. The animal sample size for each experiment was selected based on previous, well-characterized studies 47 . The number of animals in each group is specified in figure legends.
All animals were maintained in the Animal Facility of Tongji University School of Medicine. The experimental procedures involving mice were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care and Experiment Committee of Tongji University.
The surgeries and intra-articular injections for mice. We anesthetized 10-weekold male mice with ketamine and xylazine. To induce mechanical instabilityassociated OA, DMM was surgically performed by transection of the medial meniscotibial ligament in the right knee joints, as previously described 49 . Control mice received a sham operation on the knee joint.
For the time-course study, operated knee joints were collected at 0, 4, 8, or 12 weeks after surgery. For antagomir (RiboBio Tech) delivery, mice were randomized into four groups. Mice were subjected to sham operation or DMM surgery and weekly intra-articular injection of antagomir-NC or antagomir-17 (3nmol) by inserting a small needle into the sub-patellar region for three times from 1 week post-operation. For agomir delivery, mice were randomized into four groups. A total of 1.5 nmol of agomir-17 or agomir-NC (RiboBio Tech) in 8 μL of PBS was injected into the knee joints at 4 weeks after surgery. For the GDF-5 study, mice were randomized into 3 groups: 1) Sham, 2 and 3) at 4 weeks after DMM surgery, injected with 8 μL of recombinant mouse GDF-5 (100 ng; PeproTech) or the equivalent volume of vehicle (PBS) into knee joints. For antagomir delivery, mice were randomized into four groups. A combination of GDF-5 (0 or 100 ng) and 3 nmol of the antagomir (antagomir-17 or antagomir-NC, RiboBio Tech) in 8 μL of PBS was injected intra-articularly. Except where stated, agomir-, GDF-5-, and antagomir-treated mice received weekly injections for 4 weeks beginning at 4 weeks after surgery and were euthanized at 8 weeks after surgery. Knee joints were processed for histology and immunohistochemical (IHC) staining. Articular cartilage was harvested for mRNA analysis by qRT-PCR. Samples obtained from animals were excluded from further analysis if there was evidence of joint infection or if the animal died after the procedure. These exclusion criteria were established prior to the surgeries.
The cKO mice induction and operations. To activate the estrogen receptor (ER), tamoxifen (Sigma, 100 μg per gram of body weight) was intraperitoneally injected into 7-week-old miR-17~92 fl/fl and miR-17~92 cKO mice daily for 6 days 44 . To confirm knockout of miR-17, articular cartilage was isolated for qRT-PCR analysis at 1 week after tamoxifen injections. Then, sham or DMM operations were performed on 8-week-old miR-17~92 cKO mice and their miR-17~92 fl/fl littermates as described above to establish an experimental model of OA. The transgenic mice were euthanized at 4 or 8 weeks after tamoxifen injections, and knee joints were processed for histological and IHC staining. The intervention of agomir-17, agomir-19 and agomir-92 or GDF-5 in cKO mice were performed at 1 week post the initial tamoxifen injection, and analyzed at 4 weeks post-surgery.
Quantitative RT-PCR (qRT-PCR). Total RNA was extracted from cultured chondrocytes or mouse cartilage tissue with TRIzol Reagent (Life Technologies) according to manufacturer's instructions. Each in vivo cartilage sample represented pooled tissues from 2-3 knee joints. Human cartilage was homogenized with a TissueLyser-48L (Shanghai Jingxin), and RNA was isolated and purified using an RNeasy Fibrous Tissue Mini Kit (Qiagen). Then, 0.5 μg of RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent Kit (Takara), and qPCR was performed in an ABI 7500 Fast System (Applied Biosystems). GAPDH or U6 was used for normalization. The reverse transcription and PCR primers specific for U6 and the miRNAs were purchased from RiboBio, and sequences of other PCR primers used in the study were listed in Supplementary Table 1.
Histological, IHC, and FISH analyses. Mouse knee joints were fixed with 4% paraformaldehyde for 48 h, decalcified with 10% EDTA for 10 days, and dehydrated for paraffin embedding. Serial sagittal or coronal sections were harvested at 100 μm intervals to obtain 15-18 slides. The sections were cut to a thickness of 6 μm, dewaxed and stained with safranin O and fast green to visualize cartilage matrix proteoglycans or with hematoxylin and eosin (H&E) to visualize chondrocytes and matrix structures. To grade OA severity, OARSI scoring system 50 was performed by two observers blinded to the experimental groups to evaluate cartilage destruction in the medial joints. We scored the cartilage destruction from the medial tibial plateau in all DMM-involved models and medial femoral condyles in cKO mice without DMM surgery. For each joint, 3-5 sections were used for safranin O staining and the median score was representative for each biological samples. Human cartilage was cut off, fixed and embedded in paraffin and sectioned at 10-μm thickness. For IHC analysis, sections were incubated overnight at Droplet-based scRNA-seq. A total of nine littermate mice, including miR-17~92 cKO, miR-17~92 fl/fl and agomir-17-treated miR-17~92 cKO mice, were used in this experiment. Mice in the agomir-17 group received weekly injections of agomir-17 (2 nmol/injection) in the knee joints for a total of 4 injections. After mice were euthanized, the medial femoral condyle, including the articular cartilage, subchondral bone and adjacent bone marrow cavity, was dissected using scissors to ensure that intact cartilage tissue was obtained. The tissue was cut into small pieces and was then digested at 37°C for 2 h in 10 mL of DMEM containing 0.2% NB4 collagenase and 0.3 mg/mL DNase I (Thermo Scientific). The mixture was filtered through a 40-μm strainer and centrifuged at 300 × g for 10 min. Cells were counted using a hemocytometer, and~8000 cells in 25 μL of PBS per group were used for experiments. Single cells were processed on a Chromium Single-Cell Platform with a Chromium Single-Cell 3′ Library and Gel Bead Kit v2 (10X Genomics, PN-120237) and a Chromium Single-Cell A Chip Kit (10X Genomics, PN-120236) according to the manufacturer's protocol. DNA quality was assessed with an Agilent Bioanalyzer to ensure transcript integrity and purity, and amount was assessed with a Qubit fluorometric assay (Invitrogen).
Preprocessing of scRNA-seq data. Raw sequencing data were aligned to the mm10 (Ensembl 84) reference genome, and estimated cells and associated unique molecular identifiers (UMIs) were counted using the CellRanger Single-Cell Software Suite (version 2.1.1, 10X Genomics). Downstream analyses were performed with the R software package Seurat version 2.3.4 52 . First, genes expressed in <4 cells and cells expressing <200 genes were excluded. Then, the UMIs were log normalized for each cell using the natural logarithm of 1 + UMIs per 10,000. Variable genes were selected with the FindVariableGenes function using thresholds for average expression (>0.005 and <3) and dispersion (0.3). Variation in UMI counts between cells was regressed using the ScaleData function with default parameters. Principal component analysis was performed on the regressed highly variable genes with "RunPCA" (pcs.compute = 100), and the top 100 principal components were used to conduct fast Fourier transform-accelerated interpolation-based t-distributed stochastic neighbor embedding (FIt-SNE). The cells were then clustered using the function "FindClusters" (resolution = 1.2, n.start = 10, nn.eps = 0.5, k.param = 20). For each cluster, differentially expressed genes were identified using the "FindAllMarkers" or "FindMarkers" functions with default parameters. The clusters that expressed several chondrocyte markers at high levels were selected for downstream analysis. Single-cell gene expression data were analyzed for differential gene expression across the chondrocyte subsets. Differential gene expression was determined using UMI-normalized gene expression data. The Reactome, KEGG and GO Biological Functions libraries were used to analyze the scRNA-seq data. Enrichment of GO terms in the "biological process" category was conducted for differentially expressed genes (log (fold change) ≥0.5 or <−0.5) using the Bioconductor GOstats package. The top GO terms were filtered by size (between 30 and 300 genes) to select for terms that were neither excessively general nor specific.
Statistical analysis. The assessment of OARSI grades and count of immunostaining positive cells were performed in a blinded manner. Statistical significance was assessed using an unpaired, two-tailed Student's t test for comparisons between two groups to evaluate qRT-PCR data, luciferase assay data and the percentages of positive cells in tissue sections. The variance was estimated within each group of data. The equivalence of variance was confirmed using an F test for comparisons between two groups. For multigroup comparisons, one-way ANOVA followed by a post hoc test was used. For nonparametric data, the Mann-Whitney U test was used. For nonparametric, multigroup comparisons, the Kruskal-Wallis test followed by Dunn's multiple comparisons test was used. All analyses were performed with GraphPad Prism 6 software (GraphPad Software). P < 0.05 was considered statistically significant.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Single-cell RNA-seq files have been uploaded to the websites, Accession: SRX10016273, SRX10016274, SRX10016275. Raw sequencing data were aligned to the mm10 (Ensembl 84) reference genome. All the other data are available within the article and its Supplementary Information. Source data are provided with this paper. Uncropped blots are provided in Source data.